Traditional Chinese medicine composition with effect of inhibiting virus neuraminidase NA and virus 3CLpro enzyme and application of traditional Chinese medicine composition

Abstract

The invention discloses a traditional Chinese medicine composition with an effect of inhibiting virus neuraminidase NA and virus 3CLpro enzyme and application of the traditional Chinese medicine composition, and relates to the technical field of traditional Chinese medicines. The traditional Chinese medicine composition comprises a rhizoma cyperi extract and a citronella extract. The traditional Chinese medicine composition has the effects of inhibiting virus neuraminidase NA and virus 3CLpro enzyme at the same time, and the cyperus rotundus extract and the citronella extract in the traditional Chinese medicine composition can significantly improve the inhibitory activity on coronavirus main protease and influenza virus neuraminidase. Therefore, the traditional Chinese medicine composition can be matched with pharmaceutically acceptable pharmaceutic adjuvants and devices to prepare medicines or daily chemical products for prevention and / or adjuvant treatment of coronavirus or influenza virus infection.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicines, and in particular to a traditional Chinese medicine composition capable of inhibiting viral neuraminidase NA and viral 3CLpro enzyme and application thereof. Background Art

[0002] Viruses are direct risk factors that plague the quality of human survival and life, especially the recent novel coronavirus (COVID-19) and its variants such as Omicron, which cause pneumonia (new crown infection) that seriously affects people's health. Even if the vaccine is fully vaccinated, it still cannot prevent the infection and harm of virus mutations to humans. At present, chemical drugs such as ambavir monoclonal antibody / romisvir monoclonal antibody combined with biological drugs, remdesivir, Molnupiravi, Paxlovid, VV116, and traditional Chinese medicines such as Qingfei Paidu Decoction, Lianhua Qingwen Capsules, and Jinhua Qinggan Granules have been launched on the market, but they still cannot meet the prevention and control needs of the constantly mutating new coronavirus infection. The World Health Organization reminds that the new coronavirus will coexist with humans for a long time, merging with influenza viruses, making the virus more complex and diverse. Different viruses have undergone significant changes in the ways they infect humans, the targets they act on, the key enzymes of replication, and even evade the human immune system; they may also break out seasonally like influenza viruses, causing many problems in people's lives. Neuraminidase (NA) is the most important and effective therapeutic target for influenza virus. Zanamivir, peramivir, oseltamivir and lanamivir are all effective drugs targeting NA; however, due to many adverse reactions and high prices, they are not the best choice for clinical use. Therefore, it is urgent to find other highly effective and low-toxic anti-influenza virus drugs. Traditional Chinese medicine is profound and extensive. The present invention intends to provide new alternative drugs for the treatment of influenza and new coronavirus by developing a Chinese medicine composition.

[0003] Cyperus rotundus L. is a dried rhizome of the Cyperaceae plant Cyperus rotundus L. It is harvested in autumn, the hair is burned off, and it is placed in boiling water and boiled or steamed thoroughly before drying, or burned and then directly dried in the sun. It is pungent, slightly bitter, slightly sweet, and neutral; it enters the liver, spleen, and triple burner meridians; it promotes qi and relieves depression, regulates menstruation and relieves pain. It is used for liver depression and qi stagnation, chest, flank, abdominal distension and pain, indigestion, chest and abdominal distension, abdominal pain due to cold hernia, breast pain, irregular menstruation, amenorrhea and dysmenorrhea. There are currently no reports on the research of Cyperus rotundus in the fight against influenza virus and coronavirus infection, and there is no record of its inhibition of coronavirus 3CLpro enzyme and influenza virus neuraminidase (NA) activity.

[0004] Citronella is a lemongrass of the genus Cymbopogon citratus in the Poaceae family. The aboveground stems and leaves are harvested in summer and used fresh or dried for later use. It has a certain effect of repelling mosquitoes, flies and insects and reducing bacteria in wards, and is often used as an indoor fragrance. At present, there are no reports on the research of citronella in the fight against influenza virus and coronavirus infection, nor are there any records of its inhibition of coronavirus Mpro / 3Clpro and influenza virus NA activity. Summary of the invention

[0005] The purpose of the present invention is to provide a Chinese medicine composition having the function of inhibiting viral neuraminidase NA and viral 3CLpro enzyme and its application, so as to solve the problems existing in the above-mentioned prior art. The Chinese medicine composition has the function of inhibiting the activity of coronavirus main protease and influenza virus neuraminidase at the same time.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a traditional Chinese medicine composition capable of inhibiting viral neuraminidase NA and viral 3CLpro enzyme, comprising a cyperus rotundus extract and a citronella extract.

[0008] Furthermore, the Cyperus rotundus extract is a Cyperus rotundus volatile oil extract, and the Cymongrass extract is a Cymongrass volatile oil extract.

[0009] Furthermore, the mass ratio of the Cyperus rotundus volatile oil extract to the Cymbopogon citratus volatile oil extract is 1:(1-10).

[0010] Furthermore, the mass ratio of the Cyperus rotundus volatile oil extract to the Cymbopogon citratus volatile oil extract is 1:5.

[0011] Furthermore, the mass ratio of the Cyperus rotundus volatile oil extract to the Cymbopogon citratus volatile oil extract is 1:1.

[0012] The present invention also provides the use of the above-mentioned Chinese medicine composition in preparing the product described in any one of the following (1)-(6):

[0013] (1) Products that inhibit viral 3CLpro enzyme activity;

[0014] (2) Products that inhibit viral neuraminidase NA activity;

[0015] (3) Products that have the ability to inhibit both viral 3CLpro enzyme and viral neuraminidase NA activity;

[0016] (4) Products with anti-coronavirus effects;

[0017] (5) Products with anti-influenza virus effects;

[0018] (6) Products that have both anti-coronavirus and anti-influenza virus effects.

[0019] The present invention also provides a product for inhibiting the activity of viral neuraminidase NA and / or viral 3CLpro enzyme, comprising the above-mentioned traditional Chinese medicine composition.

[0020] Furthermore, the product includes medicine or daily chemical products.

[0021] The present invention also provides a product for resisting coronavirus and / or influenza virus, comprising the above-mentioned traditional Chinese medicine composition.

[0022] Furthermore, the product includes medicine or daily chemical products.

[0023] The present invention discloses the following technical effects:

[0024] Neuraminidase (NA) is the most important and most effective therapeutic target for influenza virus; coronavirus 3CLpro enzyme is a key enzyme for coronavirus replication and is the main target for the development of anti-coronavirus infection drugs; the present invention provides a Chinese medicine composition, including a cyperus rotundus extract and a citronella extract, the Chinese medicine composition has the effects of inhibiting coronavirus (inhibiting coronavirus main protease) and influenza virus (inhibiting influenza virus neuraminidase), and the cyperus rotundus extract and the citronella extract are used together to significantly improve the inhibitory activity of coronavirus main protease and influenza virus neuraminidase. Therefore, the Chinese medicine composition can be prepared into a drug (such as a nasal administration preparation, an oral spray system, etc.) or a daily chemical product (such as a hand sanitizer, a disinfectant, an emulsion, a disinfectant gel, a bath gel, a shampoo, a dishwashing liquid, a detergent, a mouthwash or a disinfectant wet wipe, etc.) for preventing and / or assisting the treatment of coronavirus or influenza virus infection with pharmaceutically acceptable pharmaceutical excipients and devices. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 This is the inhibition curve of different samples on the main protease of coronavirus. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] Example 1

[0033] 1. Preparation of volatile oil or aqueous extract

[0034] Citronella volatile oil: Take 100 g of citronella, add 8 times the weight of water, heat and reflux for 5 hours, collect the volatile oil, and obtain citronella volatile oil for later use.

[0035] Cyperus rotundus volatile oil: Take 100 g (crushed) Cyperus rotundus, add 8 times the weight of water, heat and reflux for 5 hours, collect the volatile oil, and obtain Cyperus rotundus volatile oil for later use.

[0036] Mixed volatile oil of Cyperus rotundus and Citronella: Take 50g of Citronella and 50g of Cyperus rotundus (crushed), mix, then add 10 times the weight of water, heat and reflux for 4 hours, collect the volatile oil to obtain the mixed volatile oil of Cyperus rotundus and Citronella, and set aside; filter the mother liquor to obtain the mixed water extract of Cyperus rotundus and Citronella, and set aside.

[0037] According to the same preparation method of the citronella volatile oil and the cyperus rotundus volatile oil, prepare the atractylodes lancea volatile oil, the wormwood volatile oil, the clove volatile oil, the patchouli volatile oil and the galangal volatile oil, and set aside.

[0038] 2. Enzyme Activity Inhibition Experiment

[0039] 2.1 Evaluation of inhibitory activity against coronavirus 3CLpro enzyme

[0040] In this experiment, a 3CLpro enzyme inhibitor screening kit was used to study the inhibitory activity of the test sample on Mpro / 3Clpro.

[0041] 2.1.1 Methods

[0042] (1) Preparation of test solution

[0043] Weigh 1 mg of the test sample (i.e., the volatile oil or water extract prepared in "1. Preparation of volatile oil or water extract"), add 1 mL of Assay buffer, vortex to dissolve, centrifuge at 10000 rpm for 10 min, and take the supernatant.

[0044] (2) Preparation of positive control drug

[0045] Take Ebselen (10mM, 20μL / bottle), accurately measure 10μL, add 0.99mL of Assay buffer, vortex for 30s to obtain a 100μM solution; accurately measure 200μL of the 100μM solution, add 400μL of Assay buffer, vortex for 30s to obtain a 33.33μM solution as a positive control.

[0046] (3) Mpro inhibition test

[0047] ①Assay Reagent Preparation

[0048] Take Assay buffer and Substrate and return to room temperature. According to the number of samples n, accurately measure 2019-nCovMpro / 3CL(n+3)μL, add Assay Buffer(n+3)×92μL, vortex for 30s, and store on ice.

[0049] ② Grouping and dosing

[0050] Take a black 96-well plate and add the corresponding test drugs according to the loading volume in Table 1.

[0051] Table 1

[0052] Drug testing Blank control group 100% enzyme activity group Positive control group Sample set Assay buffer 98μL 5μL —— —— Assay Reagent —— 93μL 93μL 93μL Ebselen Solution —— —— 5μL —— Drugs to be tested —— —— —— 5μL

[0053] ③Incubation

[0054] The 96-well plate after drug addition was incubated at 37°C.

[0055] ④ Add substrate and detect

[0056] Quickly add 2 μL of Substrate, mix well, place in a microplate reader, set the excitation wavelength to 340 nm, the emission wavelength to 490 nm, and measure the fluorescence value within 30 min.

[0057] (4) Calculation of inhibition rate

[0058]

[0059] 2.1.2 Results

[0060] (1) The results of the inhibitory efficacy of the test samples with a concentration of 1 mg / mL on the 3CLpro enzyme are shown in Table 2. The results show that the volatile oil of Cyperus rotundus and the mixed water extract of Cyperus rotundus + Cymongrass have a certain inhibitory effect on the 3CLpro enzyme; the volatile oil of Cyperus rotundus and the mixed volatile oil of Cyperus rotundus + Cymongrass have a better inhibitory effect on the 3CLpro enzyme.

[0061] Table 2

[0062]

[0063] (2) Percent inhibition rate of different concentrations of Cyperus rotundus samples on coronavirus 3CLpro enzyme

[0064] The Cyperus rotundus volatile oil, Cyperus cyperus volatile oil and the mixture of the two in different mass ratios prepared in the section "1. Preparation of volatile oil or water extract" were tested for the percentage inhibition rate of coronavirus 3CLpro enzyme at different concentrations. The results are shown in Table 3. The results show that at high concentrations, Cyperus cyperus volatile oil and Cyperus rotundus volatile oil can completely inhibit the 3CLpro enzyme; and the mixture of Cyperus cyperus volatile oil and Cyperus rotundus volatile oil has better activity.

[0065] Table 3

[0066]

[0067] Note: The concentrations in the table are those of Cyperus rotundus volatile oil, Cymbopogon citratus volatile oil or a mixture of the two.

[0068] The inhibition curve was drawn with the logarithm of sample concentration as the horizontal axis and the inhibition rate as the vertical axis (see Figure 1), and the data were processed by GraphPad Prism8.0 software. The results showed that both volatile oils of Cyperus rotundus and Citronella cymbopogon had good inhibitory activity on 3CLpro enzyme, and the half inhibition concentration of Citronella cymbopogon volatile oil IC50 was 628.4 μg / mL; the two volatile oils were mixed in different proportions to produce a synergistic effect, and the half inhibition concentration of Citronella cymbopogon volatile oil was significantly reduced to 358.1 μg / mL; when the content of the two volatile oils reached 3000 μg / mL, the activity of 3CLpro enzyme could be completely inhibited. At the same dose, the combined use of volatile oils of Cyperus rotundus and Citronella cymbopogon had a better inhibitory effect.

[0069] 2.2 Evaluation of inhibitory activity against influenza virus neuraminidase 2.2.1

[0071] In this experiment, a neuraminidase inhibitor screening kit was used to study the inhibitory activity of the test sample (i.e., the volatile oil or aqueous extract prepared in the "1. Preparation of volatile oil or aqueous extract") on influenza virus neuraminidase. The positive control drug was Oseltamivir, and the detection method was referenced to 2.1. 2.2.2

[0073] (1) The results of the inhibitory efficacy of the test samples with a concentration of 1 mg / mL on influenza virus neuraminidase are shown in Table 4. The results show that cymbopogon volatile oil and the mixed water extract of cyperus rotundus + cyperus rotundus have a certain inhibitory effect on influenza virus neuraminidase; cyperus rotundus volatile oil and the mixed volatile oil of cyperus rotundus + cyperus rotundus have a better inhibitory effect on influenza virus neuraminidase.

[0074] Table 4

[0075]

[0076] (2) Percent inhibition rate of influenza virus neuraminidase by samples of different concentrations

[0077] The Cyperus rotundus volatile oil, Cyperus cyperus volatile oil and the mixture thereof in different mass ratios prepared in the section "1. Preparation of volatile oil or water extract" were tested for the percentage inhibition rate of influenza virus neuraminidase at different concentrations. The results are shown in Table 5. The results show that at high concentrations, Cyperus cyperus volatile oil and Cyperus rotundus volatile oil can better inhibit influenza virus neuraminidase; and the mixture of Cyperus cyperus volatile oil and Cyperus rotundus volatile oil produces a synergistic effect and has better activity.

[0078] Table 5

[0079]

[0080] Note: The concentrations in the table are the concentrations of Cyperus rotundus volatile oil, Cyperus cymophylla volatile oil, the mixture of Cyperus rotundus volatile oil and Cyperus cymophylla volatile oil, or the mixed water extract of Cyperus rotundus + Cyperus cymophylla.

[0081] Example 2

[0082] Take 1 mL each of the Cyperus rotundus volatile oil and the Cymbopogon citratus volatile oil prepared in Example 1, add 2.0 mL of Tween 80, mix well, then add 5 g of glycerol, 1 g of squalane, 1 g of sodium hyaluronate, 0.03 g of sodium propylparaben and 1.5 g of potassium sorbate, and finally add purified water to 100 mL, put into a spray bottle, and obtain an alcohol-free disinfectant.

[0083] Example 3

[0084] Take 1 mL each of the Cyperus rotundus volatile oil and the citronella volatile oil prepared in Example 1, add 2.0 mL of Tween 80, mix well, then add 15 g of glycerol, 5 g of squalane, 3 g of sodium hyaluronate and 0.5 g of menthol, and finally add purified water to 100 mL, sterilize, and put into a dropper bottle to obtain alcohol-free nasal drops.

[0085] Example 4

[0086] Take 0.1 g of the volatile oil of Cyperus rotundus and 0.1 g of the volatile oil of Citronella prepared in Example 1, add 1.10 g of Tween 80, 1.0 g of the aromatic refreshing agent peppermint oil and 2 g of xylitol, mix well, add sweetener, and add purified water to 100 mL to make an alcohol-free mouthwash.

[0087] Example 5

[0088] Take 5g of carbomer, add 25g of purified water, fully swell, take 1.0g of the volatile oil of Cyperus rotundus and volatile oil of Citronella prepared in Example 1, mix them evenly, add 1.0g of Tween 80 to dissolve, then add 25g of glycerol, 0.5g of aromatic refreshing agent peppermint oil, 2g of xylitol and 3g of triethanolamine, add 0.02g of nanosilver, and finally add purified water to 100mL to obtain a carbomer gel.

[0089] Example 6

[0090] Take 0.2 mL of the Cyperus rotundus volatile oil prepared in Example 1 and 1.0 mL of the Cymbopogon citratus volatile oil prepared in Example 1, add 1.2 mL of Tween 80, mix well, add 15 g of glycerol, 5 g of squalane, 3 g of sodium hyaluronate and 0.05 g of chlorhexidine acetate, and finally add 70% by volume ethanol aqueous solution to 100 mL to obtain a quick-drying spray hand disinfectant.

[0091] Example 7

[0092] Take 1 g of the volatile oil of Cyperus rotundus and 1 g of the volatile oil of Citronella prepared in Example 1, add 0.4 g of the mixed aqueous extract of Cyperus rotundus + Citronella prepared in Example 1, 2 g of Tween 80, 10 g of glycerol, 5 g of squalane, 5 g of carbomer, 30 g of deionized water, 50 g of ethanol, 3 g of sodium hyaluronate, 8 g of emollient, 0.2 g of benzalkonium bromide and 0.5 g of triethanolamine until the solution becomes a gel state to prepare a wash-free hand sanitizer.

[0093] Example 8

[0094] Take 10g each of Cyperus rotundus and fresh Cymbopogon citratus, add 200mL of water and soak for 30 minutes, reflux to extract volatile oil for 4 hours, collect the volatile oil and aqueous solution, then add 1g of xylitol, 1g of rattan tea extract, and 0.2mL of clove volatile oil as a flavoring agent, and make 100mL of mouthwash according to the conventional method, which has good breath freshening and antibacterial and anti-inflammatory effects.

[0095] Example 9

[0096] Take 1 g each of the Cyperus rotundus volatile oil and the citronella volatile oil prepared in Example 1, add 0.1 g of sodium carboxymethyl cellulose, 5 g of ethanol, 0.5 g of menthol, 1 g of citric acid, 1 g of sorbitol, 1.5 g of xylitol and about 0.5 g of apple flavor, strawberry flavor and orange flavor, make the volume to 1000 mL with purified water and stir evenly to prepare a mouthwash.

[0097] Example 10

[0098] Take 20g of stearic acid, 5g of glyceryl monostearate, 10g of liquid paraffin and 1.5g of vaseline, heat in a water bath until dissolved, and continue to heat to 70°C; separately take 2g of sodium hydroxide and 0.2g of ethylparaben, add distilled water to 100g, heat to 70°C, slowly pour in oil phases such as stearic acid, stir while adding, until emulsification is complete, cool to 45°C, then drip 1.0g each of the volatile oil of Cyperus rotundus and the volatile oil of Citronella prepared in Example 1, and 0.2g of peppermint oil as a fragrance, and mix well to obtain a fragrant antibacterial and antiviral emulsion.

[0099] Embodiment 11

[0100] Take 300g of stearyl alcohol, 250g of white vaseline, 15g of sodium lauryl sulfate, 120g of glycerol, 10g of triethanolamine, 3g of the volatile oil of Cyperus rotundus prepared in Example 1, and 3.0g of the volatile oil of Cyperus rotundus prepared in Example 1; add 0.3mL of peppermint oil, 0.5mL of Angelica dahurica extract, 0.4mL of rose essential oil, and 1g of antibacterial agents ethylparaben and 0.5g of chlorhexidine acetate, and add distilled water to 1000g to prepare a fragrant antibacterial and antiviral vanishing cream.

[0101] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A Chinese medicine composition having the function of inhibiting viral neuraminidase NA and viral 3CLpro enzyme, characterized in that: Including Cyperus rotundus extract and Cyperus rotundus extract.

2. The Chinese medicine composition according to claim 1, characterized in that: The cyperus rotundus extract is a cyperus rotundus volatile oil extract, and the citronella extract is a citronella volatile oil extract.

3. The Chinese medicine composition according to claim 2, characterized in that: The mass ratio of the Cyperus rotundus volatile oil extract to the Cymbopogon citratus volatile oil extract is 1:(1-10).

4. The Chinese medicine composition according to claim 3, characterized in that: The mass ratio of the Cyperus rotundus volatile oil extract to the Cymbopogon citratus volatile oil extract is 1:

5.

5. The Chinese medicine composition according to claim 3, characterized in that: The mass ratio of the Cyperus rotundus volatile oil extract to the Cymbopogon citratus volatile oil extract is 1:

1.

6. Use of the Chinese medicine composition according to any one of claims 1 to 5 in the preparation of the product according to any one of the following (1) to (6): (1) Products that inhibit viral 3CLpro enzyme activity; (2) Products that inhibit viral neuraminidase NA activity; (3) Products that have the ability to inhibit both viral 3CLpro enzyme and viral neuraminidase NA activity; (4) Products with anti-coronavirus effects; (5) Products with anti-influenza virus effects; (6) Products that have both anti-coronavirus and anti-influenza virus effects.

7. A product for inhibiting viral neuraminidase NA and / or viral 3CLpro enzyme activity, characterized in that: A Chinese medicine composition comprising any one of claims 1 to 5.

8. The product according to claim 7, characterized in that The products include medicines or daily chemicals.

9. A product for resisting coronavirus and / or influenza virus, characterized in that: A Chinese medicine composition comprising any one of claims 1 to 5.

10. The product according to claim 9, characterized in that The products include medicines or daily chemicals.

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